Embryonic stem (ES) cells are pluripotent cells derived from embryos and which have the ability to differentiate into all three germ layers. In an embryo, such cells contribute to the formation of all tissues and organs of the organism, including the germ cells.
ES cells express a number of specific molecular markers, such as Oct4, Nanog, Sox2, Rex1 and alkaline phosphatase. ES cells also express a number of specific cell surface markers, such as SSEA-1, SSEA-2, TRA 1-60, and TRA 1-81. A further characteristic of undifferentiated ES cells is cytoplasmic organization of F-actin, with associated non-muscle myosin. In undifferentiated ES cells F-actin is organized in periphery organized cortical rings, where cells make direct contact with each other.
The main source of ES cells is the embryoblast, the inner pluripotent component of pre-implantation embryos. In mammals, such as mice and other rodents, primates and humans, this pluripotent component is referred to as the inner cell mass (ICM), because the cells are organised as a clump of pluripotent cells within the early embryo. In ungulates, this component is called the embryonal disk (ED), because the cells of this component are organised as a flattened, mostly one-layered structure.
The research and medical applications of ES cells are well recognised. However, these applications have been hampered by the inability and/or inefficiency of methods of isolation of ES cells from nearly all species.
For example, the efficiency of isolation of ES cells, and the establishment of ES lines, is low. Even for mouse strains that are routinely used to establish ES lines, the efficiency of isolation does not normally exceed 2 to 3% of the embryos used.
The methods of isolating ES cells also generally require a step of separating the pluripotent component from other non-pluripotent cells, such as trophocectoderm and extra-embryonic endoderm, in order to reduce the overgrowth of these other cells. These methods of separating the pluripotent component from other cells usually involve either an immunosurgical step or a step involving the mechanical removal of trophectoderm cells from the inner cell mass.
In this regard, it has also not been possible to isolate pluripotent cells from whole embryos without some form separation of the pluripotent component away from other cells in the embryo.
In addition, for most mammals the establishment of true ES cell lines, in terms of their ability to participate in formation of all organs and tissues (including germline lineages) of chimeric animals, has not yet been reported.
Although there may be a number of reasons for the inability and/or inefficiency for isolating ES cells, it is likely that the small number of cells that are pluripotent in the embryo, and the inability to maintain pluripotent cells isolated from an embryo in an undifferentiated state, contribute to this inability and/or inefficiency.
The present invention relates to a method of isolating pluripotent cells from embryos that does not require separation of the pluripotent component from other cell types and which can also be used to isolate pluripotent cells from whole embryos directly.
A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.